stimulated the growth of some intracellular bacteria. Other groups have shown a clear stimulation of parasite growth with cytokines.6.7 On the basis of these observations, and on the emerging clinical support for their importance to man, we urge caution in the widespread application of cytokine-based immunotherapy, especially for immunocompromised patients. Whether antibiotic cover will be sufficient to prevent these infectious complications remains to be established. Pulmonary Research Unit, Centre Hospitaller Universitaire de Sherbrooke, Sherbrooke J1 H 5N4, Canada, and 19 Buckingham Gate, London SW1, UK
M. DENIS E. O. GREGG
Jablons D, Bolton E, Martins S, et al. Interleukin-2 based immunotherapy alters circulating neutrophil Fc receptor expression and chemotaxis. J Immunol 1990,
Klempner MS, Noring R, Mier JW, Atkins MB An acquired chemotactic defect in neutrophils from patients receiving interleukin-2 immunotherapy. N Engl J Med
We have investigated twice as many ulcerative colitis patients did Murch et al, who quantified endothelin-1 by radioimmunoassay. The presence of a similar amount of endothelin-1 in colonic mucosa of patients with active ulcerative colitis and in controls challenges the hypothesis that endothelin-1 may have a role in the pathogenesis of ulcerative colitis. Thus, the hypothesis, although highly attractive, should be tested further before definite conclusions can be drawn.
1990; 332: 959-65 3. Denis M, Campbell D, Gregg EO. Interleukin-2 and granulocyte-macrophage colony stimulating factor stimulate growth of a virulent strain of Escherichia coli Infect Immun 1991; 59: 1853-56. 4. Denis M, Gregg EO Recombinant tumour necrosis factor-alpha decreases whereas recombinant interleukm-6 increases growth of a virulent strain of Mycobacterium avium in human macrophages Immunology 1990, 71: 139-41. 5. Denis M, Gregg EO. Identification of cytokines which enhance (CSF-1, IL-3) or restrict (IFN&ggr;) growth of intramacrophage Listeria monocytogenes. Immunol Lett 1991; 27: 237-42. 6 Mazingue C, Cottrez-Detoeuf F, Louis J, et al. In vitro and in vivo effects of interleukin-2 on the protozoan parasite Leishmania Eur J Immunol 1989; 19: 487-92 7.
Kongshavin PAL, Ghadirian E Enhancing and suppressive effects of tumor necrosis factor/cachectin on Trypanosoma musculi growth Parasite Immunol 1989, 10: 581-87.
Colonic endothelin-1 immunoreactivity in active ulcerative colitis SIR,-We have also investigated Dr Murch and colleagues’ hypothesis (Feb 15, p 381) that endothelin-1, a potent vasoconstrictor, may have a role in the pathogenesis of inflammatory bowel disease. Colonoscopic biopsies were done in nine patients with active ulcerative colitis, three patients with ulcerative colitis in remission, and eight healthy controls. All ulcerative colitis patients were receiving only sulphasalazine at the time of biopsy. Controls had colonoscopy for rectal bleeding due to haemorrhoids. All patients underwent flexible sigmoidoscopy (Olympus CFP10S sigmoidoscope) of the distal 50 cm of the colon. Biopsy specimens were obtained from an area 10-15 cm above the anal verge in controls and in patients in remsision, and from sites of active disease in patients with active ulcerative colitis. Immediately on excision biopsy specimens were frozen in liquid nitrogen and afterwards weighed and homogenised in 10 volumes of 2 molar acetic acid containing 10 mmol/1 mercaptoethanol and phenylmethylsulfonylfluoride (1 ug/ml). The homogenate was centrifuged at 4°C. The supernatant was kept and the pellet resuspended and centrifuged. Supernatants were combined and kept at - 20°C until lyophilised. After lyophilisation the protein was resuspended in borate buffer, pH 84.
Endothelin-1 was measured by radioimmunoassay (Nichols Institute Diagnostics, Netherlands). Endothelin-I was quantified by a competitive radioimmunoassay with radioiodinared tracer and polyclonal antibody. Bound and free protein were separated by a second antibody solid-phase system, followed by centrifugation; sensitivity of the assay was 1 pg/ml and crossreactivities were: endothelin-I 100%, endothelin-2 52%, endothelin-3 96%, big endothelin 7%, angiotensin II less than 0-1%, corticotropin less than 0-1%, and vasopressin less than 0 1 %. Differences between the various groups were evaluated by Student’s t test for unpaired data and confirmed by the Mann-Whitney test. Endothelin-1was detected in colonic mucosa of controls, as well as in patients with ulcerative colitis. Mean endothelin-1 content in colonic mucosa of controls was 5-7 (SEM 0-3) pg/mg wet weight tissue. Mean endothelin-I content in inflamed colonic mucosa of nine patients with active ulcerative colitis was 4-7 (0-6) and did not differ from that in colonic mucosa of controls. In three ulcerative colitis patients in remission, mean endothelin-I content was 2-9 (0-8) pg/mg wet tissue.
DANIEL RACHMILEWITZ RAMI ELIAKIM ZVI ACKERMAN FANNY KARMELI
Department of Medicine, Hadassah University Hospital, PO Box 24035, Jerusalem 91240. Israel
SIR,-In the European Community about 20 million tonnes of derived basically from mineral oil, are needed annually, much of it destined for the rubbish-bin. Less
considerations have led
development of completely biodegradable "plastic" coatings derived from gluten. These gluten-derived packagings seem likely to replace plastic material on the Dutch market and in the rest of Europe too. The Dutch Horticultural Society plans to provide our fruits and vegetables with an edible gluten-derived coating, the launching of which awaits the results of experiments in Germany. It is not obligatory to label gluten additives in foodstuffs for sale. Increasing the dietary gluten content of a whole population without a clear labelling system may to
have several consequences. The Dutch Coeliac Disease Society estimates the prevalence of biopsy-proven coeliac disease in the Netherlands to be 12 per 100 000, a rate that is lower than that in neighbouring countries but which is increasing rapidly. The low rate is at least partly due to underdiagnosis,l which means that people in the Netherlands with as yet undiagnosed coeliac disease will incur extra risks if the dietary gluten content increases. A life-long strict gluten-free diet is recommended in coeliac disease and the ingestion of very small amounts of gluten, even without the accompaniment of clinical or serological responses, induces changes that are detectable morphometrically.3 For the sake of all recognised gluten-sensitive Europeans-and gluten-sensitive Europeans yet to be identified-a united European approach is needed to keep gluten pollution at a distance. This should include giving gluten an E-number with an obligation to label. Department of Hepatogastroenterology, Rijnstate Hospital, Arnhem
TIM L. TH. A. JANSEN
Dutch Coeliac Disease Utrecht
LOES KÖHLER PETER H. Z. KARSSEN CYP G. J. WAGENAAR
Department of Hepatogastroenterology, 6814 HK, Arnhem, Netherlands
CHRIS J. J. MULDER
Jansen TLTA, Wagenaar CGJ, Mulder CJJ. Coeliac disease in the Netherlands. In. Mearin ML, Mulder CJJ, eds. Coeliac disease: 40 years gluten-free. Dordrecht. Kluwer, 1991. 169-79. 2. Holmes GKT, Prior P, Lane MR, et al. Malignancy in coeliac disease effect of a gluten-free diet. Gut 1989; 30: 333-40. 3 Mayer M, Greco L, Troncone R, et al. Compliance of adolescents with coelic disease with a gluten free diet. Gut 1991, 32: 881-85. 1.
Three-dimensional fetal ultrasound SIR,-A conventional ultrasound scan presents a difficult challenge to those seeking a three-dimensional image. However, the requirements1 can be met by adapting existing equipment. A compact position sensor attached to the probe provides continuous spatial location tracking without interfering with the freehand technique of the ultrasonographer. This electromagnetic device (Polhemus Inc) has a source generator and sensor receiver both with three orthogonal coils and provides position on x, y, and z axes to within 0 25 mm and angular orientation accurate to about 02°.-’A variety of transducers may be used. A simple calibration procedure is needed to provide registration with different transducer/display
Fig 1-3D ultrasound
of 8-week fetus.
Upper (3D image)- amber represents true surface, grey scale cut surface Yolk sac sits to left of fetal head Groove of cranial midline ectodermal cleft, limb buds, and cut surfaces of umbilical cord are apparent Proximal protuberance of umbilical cord represents returning midgut migration Lower (schematic representation) 1 =cutsurfaceofuterus,2=head, 3=ectodermal cleft, 4= yolk sac, 5 R upper limb bud, 6 = R lower limb bud, 7=protuberance of proximal umbilical cord representing returning midgut migration =
combinations. Bulky ferromagnetic metal objects have to be kept away from the source-sensor device. For three-dimensional ultrasonic imaging of the fetus scanning can be done as usual with transabdominal 35 MHz or transvaginal 50 MHz transducer. Most manufacturers do not provide access to digital data within their scanners so data are recorded on video as two-dimensional slices. Digitisation of the video signal at a real-time frame rate is straightforward and this double conversion results in very little deregulation of normal ultrasound images. Several hundred slices at 256 x 256 resolution can be stored but typically 60-80 slices in a region of interest have to be stored. The acquisition sweep usually takes a few seconds and can be repeated immediately if, for example, fetal movement occurs. We have developed a multiprocessor special purpose workstation for interactive acquisition and display of three-dimensional data.4,s
Software has been developed to enhance the display by depth and surface shading, for example, and overlying data can be removed by image surgery, revealing otherwise obscured surfaces. The ultrasound machine we use is an Acuson 128 and early fetal
Fig 2-3D ultrasound scan of 18-week fetus. Upper (3D image) facial structure and appendages well defined. Lower (schematic representation). 1 = cut surface of uterus, 2 = mouth, 3 = chest wall, 4= L upper arm, 5 = lower legs anatomy is best seen with a 5 MHz transvaginal probe. (Further technical details are beyond the scope of a letter but are obtainable from 1. M. G. K.) Fig 1 shows an 8-week fetus. This 3D representation shows all four developing limb buds. The proximal umbilical bulge representing the extraembryonic umbilical midgut migration is also prominent. Image surgery to the surrounding uterus and placenta, with rotation, gives a full circumferential view allowing appreciation of the midbrain and hindbrain flexures. The midline cranial ectodermal cleft is well visualised. The yolk sac is still obvious but the loop of umbilical cord has been "resected" for display purposes. The cut ends of the efferent and afferent cord to the sac and returning mid-gut migration are also easily seen as small black discs. Transabdominal scanning with a 3-5 MHz probe of an 18-week fetus (fig 2) shows good detail to the face, chest wall, and limbs. The main benefit of this technique is to give collective coherence to a series of 2D slices which individually would, at an early stage, have little meaning otherwise. Recognition of fetal abnormalities is
handicapped by our poor ability to recognise structure, attitude, and shape. Furthermore, fetal volume estimation by 3D ultrasound is
also feasible and could lead to improved methods of and fetal weight estimation. Department of Diagnostic Imaging, Middlesex Hospital, London W1N 8AA, UK, and Medical Physics Department, University College Hospital, London
I. M. G. KELLY
J. E. GARDENER W. R. LEES
WR, Gardener JE, Gillams A. Three-dimensional ultrasound of the fetus. Radiology 1991; 181(P): 132. 2. Krieg JC, Graeme RJ. Three-dimensional echocardiography, initial feasibility testing: biomedical engineering report BCR 20006.003. Colchester, Vermont: Polhemus Navigation Sciences, 1983. 3. Krieg JC, Graeme RJ. Laboratory measurements to determine distortion due to an echocardiographic doppler transducer: biomedical engineering report BCR 20006.004A. Colchester, Vermont: Polhemus Navigation Sciences, 1983. 4. Gardener JE. Three-dimensional imaging of soft tissues using ultrasound. In: 3D imaging for medicine. (IEE Colloq Dig 91/083). London: Institute of Electrical Engineers, 1991. 5. Tan AC, Richards R. Developing the MGI (medical graphics imaging) workstation: a multitransputer based medical graphics system. In: Transputer 1991. Amsterdam: 1. Lees
IOS Press, 1991: vol II, 801-12.
We thank Mrs M. Sengteller for technical assistance, and Y. Nakamura (Tokyo), P. Meera Khan (Leiden), and R. White (Salt Lake City) for providing DNA probes and primer sequences. This study was supported by
Misleading phenotype in kindred with familial adenomatous polyposis SIR,-Familial adenomatous polyposis (FAP) is caused by
the Deutsche Krebshilfe. a
mutation of the APC-gene on chromosome 5q21.1,2 Both direct and indirect genotype analyses depend on the reliable definition of the phenotype. We report a family with FAP in which physical examination of a person at risk and linkage analysis led to different conclusions. The mother (1/2, figure) died at age 36 years from unresectable desmoid tumours; her brother (1/3) died at age 33 from carcinoma of the rectum with liver metastases. 9 years after the mother’s death FAP was diagnosed in the daughter (11/1) because of multiple polyps in the colon and the stomach, osteomas and exostoses of the jaw, and facial sebaceous-cyst-like tumours-a condition described as Gardner syndrome (GS). She had colectomy but died soon afterwards at age 27 from unresectable desmoid tumours. Her brother (11/3) also had GS. He had a maxillary osteoma, numerous soft tissue tumours, and polyposis of the colon. He had colectomy in 1988 and is well at age 32. Repeated endoscopic examinations failed to detect colonic polyps in another brother (11/2). Nevertheless, in this man, who is now aged 33, FAP was presumed because of numerous soft-tissue tumours on arms, legs, and abdominal wall. Furthermore, fundoscopy revealed a total of three hyperpigmented spots in the retinas of both eyes, suggestive of FAP. In this family the mutation at the APC locus is not yet established. Indirect genotype analysis, however, showed that 11/2 and 11/3 have inherited the same paternal but different maternal haplotypes defined by markers flanking the APC locus (figure). Non-paternity was excluded and a double recombination is very unlikely (probability < 1 in 10 000). Thus, according to indirect genotype analysis FAP will probably not develop in 11/2, since locus heterogeneity for FAP has not been reported.
flanking the APC
Histological examination of soft-tissue tumours revealed typical lipomas in 11/2, whereas in 11/3 the tumours were lipomas, fibromas, and epidermoid cysts. In his original description Gardnerreported fibromas and epidermoid cysts as skin manifestations of the syndrome. Later, lipomas or soft tissue tumours in general were claimed to be part of the syndrome.4,5 In view of our observation, lipomas alone are not sufficient for presymptomatic diagnosis of FAP in individuals at risk. Congenital hypertrophy of the retinal pigment epithelium (CHRPE) was reported to be a sensitive indicator of FAP, especially when seen bilaterally, as in 11/2. Small retinal pigmentations, however, are not uncommon in the general population. Therefore, further investigations are needed to establish their diagnostic specificity. The independent segregation of extracolonic symptoms suggestive of FAP and the DNA markers flanking the APC region could also be explained by genetic heterogeneity. More likely, extracolonic features in individuals at risk are not always reliable presymptomatic markers for FAP; thus, indirect genotype analysis based on extracolonic manifestations of FAP should be interpreted with caution.
Institut fur Humangenetik der Universitat, D-5300 Bonn 1, Germany
REINER CASPARI WALTRAUT FRIEDL PETER PROPPING
1. Kinzler KW, Nilbert MC, Su LK, et al. Identification of FAP locus genes from chromosome 5q21. Science 1991; 253: 661-65. 2. Groden J, Thliveris A, Samowitz W, et al. Identification and characterization of the familial adenomatous polyposis coli gene. Cell 1991; 66: 589-600. 3. Gardner EJ, Richards RC. Multiple cutaneous and subcutaneous lesions occurring simultaneously with hereditary polyposis and osteomatosis. Am J Hum Genet 1953; 5: 139-47. 4. Collins DC. The frequent association of other body tumors with familial polyposis. Am J Gastroenterol 1959; 31: 376-81. 5. Pierce ER, Weisbord T, McKusick VA. Gardner’s syndrome: formal genetics and statistical analysis of a large Canadian kindred, Clin Genet 1970; 1: 65-80. 6. Traboulsi EI, Krush AJ, Gardner EJ, et al. Prevalence and importance of pigmented ocular fundus lesions in Gardner’s syndrome. N Engl J Med 1987; 316: 661-67. 7. Polkingthorne PJ, Ritchie S, Neale K, et al. Pigmented lesions of the retinal pigment epithelium and familial adenomatous polyposis. Eye 1990; 4: 216-21
Calcitonin and bone diseases SIR,-Although we are aware that in some countries, especially in Italy, drug consumption and drug prescription are not always based on scientific evidence of efficacy, we are afraid that Dr Magrini and colleagues (Feb 22, p 499) have misjudged or misunderstood the role of calcitonin in bone diseases. Calcitonin is a polypeptidic hormone known for almost 30 years to inhibit osteoclast activity. It binds to high-affinity receptors on osteoclasts.1Although the physiological role of calcitonin in man has not yet been fully elucidated, many studies have shown that it has beneficial effects on the clinical and biological disturbances of diseases characterised by excessive bone remodelling. In postmenopausal women, calcitonin has at least three welldemonstrated activities. Four studies record the use of calcitonin as an alternative to oestrogen replacement therapy in the prevention of postmenopausal osteoporosis.1’ A reduction of lumbar bone loss equal to that obtained with oestrogen was achieved with a daily regimen of 5000 IU human calcitonin over 2 years, equivalent to about 50 IU per week in healthy postmenopausal women using self-administered subcutaneous injections.’ We published the first report on the efficacy of nasal calcitonin in preventing postmenopausal bone loss.2 This short-term, open, randomised controlled study was extended to 3 years and the results after this time confirmed these preliminary data.6 A preventive effect of higher doses (100 IU per day) of nasal salmon calcitonin on trabecular bone loss in the first years of natural menopause was also demonstrated in two double-blind studies.3,4 Several workers have shown a positive effect of calcitonin on established osteoporosis. Injection of calcitonin is effective in the treatment of postmenopausal osteoporosis both in short-term, uncontrolled studies and in long-term, placebo-controlled studies,?,8Reported results of long-term injections of calcitonin in